Field of the Invention
Small molecule ligands that bind to sites on the large extracellular loop of human CD81 were identified using structure-based methods. Ligands that block or interfere with attachment, invasion and infection of cells by Plasmodium parasites were produced and identified. Conjugates of these ligands were made by linking together two or more of these ligands or their CD81-binding moieties. Such conjugates can bind to different sites on CD81 and exhibit a greater specificity and a higher affinity for CD81 that can the individual ligands. These ligands and ligand conjugates can inhibit the binding of Plasmodium or inhibit the interaction of Plasmodium with host cells expressing CD81, such as human hepatocytes.
Description of the Related Art
Malaria, a disease transmitted by the Anopheles mosquito vector, has been a subject of research interest since the 1880's and remains one of the most important global diseases. Malaria caused by Plasmodium falciparum has existed for 50,000-100,000 years but the incidence of human malaria infection likely increased about 10,000 years ago with the adoption of agricultural methods associated with pooled water where mosquitos breed (1). Malaria antigen has been detected in skin and lung samples of Egyptian mummies in 3200 and 1340 BC (5, 6). The causative agent of malaria was discovered by a scientist called Laveran who discovered it in 1884 after examining the blood smears from infected patients (2, 3). In 1898, Scottish physician Sir Ronald Ross revealed the complete life cycle of malaria and proved its vector was a mosquito. In 1902 he was awarded the Nobel Prize based on his work (4).
Malaria is caused by protozoan parasites belonging to the genus Plasmodium. Human malaria infection is commonly caused by four different species which are Plasmodium malariae, Plasmodium ovale, Plasmodium vivax and Plasmodium falciparum, with P. falciparum and P. vivax being the predominant global species causing malaria (7).
Malaria infection is initiated by the introduction of the parasite into a human by mosquito bites and subsequent invasion of the sporozoite stage of the parasite into the hepatocytes (liver stage) which release the merozoite state of the parasite which invades red blood cells (blood stage), Hepatocytes express CD81 a determinant used by the malaria parasite to invade them. In order for Plasmodium parasites to propagate in the host and establish an infection, they are internalized into the hepatocytes (8). The mechanism of interaction of Plasmodium sporozoites with hepatocytes is still not clear however, human CD81 was found to be one of the significant surface proteins on the hepatocytes required by the sporozoites to invade the liver cells (9-11).
CD81 belongs to the tetraspanin family. It possesses or confers functions such as cell adhesion, migration, cell fusion, co-stimulation, signal transduction, and differentiation (12). CD81 silencing by anti-CD81 antibodies substantially inhibits the infection of hepatocytes by Plasmodium falciparum sporozoites. Additionally, it was found that Plasmodium yoelii sporozoites do not have the ability to infect CD81-deficient mouse hepatocytes both in vitro and in vivo (9-11).
Yalaoui et al. (13) identified specific amino acid residues in CD81 that are important for Plasmodium infection by conducting mutational studies. They found a stretch of 21 amino acid residues were required for invasion (135-VVDDDANNAKAVVKTFHETLD-155) (FIG. 1) (13)(SEQ ID NO: 5). Despite many prior advances there remains a profound need for new ways to prevent and treat malaria.